The broad objective of this proposal is to identify and characterize mammalian gene regulatory networks controlled by endogenously expressed double-stranded RNA (dsRNA) and to determine their function. DsRNA triggers a number of different gene silencing processes commonly referred to as RNA silencing. The identification of dsRNA triggers of gene silencing and the elucidation of their functions is a complex task because dsRNA is produced from many different sources (repetitive gene loci, miRNA genes, sense/antisense overlapping mRNA transcripts) in a tissue-specific and developmentally regulated manner. In retrospect, it is surprising that such a fundamental mode of genetic regulation has been overlooked. Comprehensively collecting the sequence information of RNA silencing regulators and targets will provide new starting points for human genetic studies aimed at characterizing the factors contributing to multigenic or idiopathic diseases. The analysis will be a team effort that brings together four laboratories interested in understanding the mechanisms and function of RNA silencing as a fundamental gene regulatory mechanism. These laboratories are specialized in biochemistry, computational biology, immunology and metabolic disease. The specific aims of the proposed project are: 1. Comprehensive recording of small RNA profiles of mammalian cell lines and tissues. 2. Development of computational and experimental methods for the identification and validation of targets of RNA silencing and RNA silencing regulatory networks. 3. Cell culture and in vivo functional characterization of RNA silencing regulatory networks using classic and conditional knockout technology in mice, in particular those related to the development of the immune system and the pancreas, as well as computational modeling of these networks. 4. Linkage analysis of genetic variation in RNA silencing regulatory networks in type 2 diabetes, supported by a relational database of small RNAs and their respective mRNA target sites available to the medical and general scientific community.